Malignant hyperthermia (MH) and central core disease (CCD) arise from mutations in the skeletal muscle[unreadable] ryanodine receptor (RyR1). Although MH and CCD mutations in RyR1 alter mechanical coupling between[unreadable] sarcolemmal dihydropyridine receptors (DHPRs) and opposing Ca2+ release channels of the sarcoplasmic[unreadable] reticulum (SR), the integrated effects of these mutations on multiple subcellular Ca2+ transport processes are[unreadable] poorly understood. The long-term goal of this project is to determine the cellular/molecular mechanisms by[unreadable] which MH and CCD mutations in RyR1 alter Ca2+ signaling interactions between the sarcolemma, SR, and[unreadable] mitochondria (the "Ca2+ signaling triad"). Specifically, this project will test the hypothesis that "MH/CCD[unreadable] mutations in RyR1 enhance excitation coupled Ca2+ entry (ECCE) activity, sensitize voltage- & ligand-gated[unreadable] SR Ca2+ release, and alter mitochondrial Ca2+ uptake during EC coupling." Aim #1 will[unreadable] characterize effects of several common RyR1 MH/CCD mutations on bi-directional DHPR-RyR1 coupling in[unreadable] skeletal myotubes and fully differentiated muscle fibers derived from MH knock-in mice generated by Core B.[unreadable] Aim #2 will test if MH/CCD mutations in RyR1 elevate steady-state resting Ca2+ by promoting a depletion of[unreadable] SR Ca2+ and increasing the activity of sarcolemmal ECCE channels. Experiments will use SR-targeted, Ca2+-[unreadable] sensitive fluorescent "cameleons" to directly report changes in SR Ca2+ and whole-cell patch clamp[unreadable] measurements to monitor changes in ECCE activity. Aim #3 will determine the degree to which mitochondrial[unreadable] triad targeting and local SR-mitochondrial Ca2+ signaling is altered by MH/CCD mutations in RyR1.[unreadable] Experiments in collaboration with Core D will use electron microscopy to assess mitochondrial morphology,[unreadable] localization, and triad targeting in FOB fibers of normal and MH/CCD knock-in mice. Functional experiments[unreadable] will use confocal microscopy, high-speed Ca2+ imaging and mitochondrial-targeted ratiometric pericam to[unreadable] report effects of MH mutations on the magnitude, kinetics, and voltage-dependence of mitochondrial Ca2+[unreadable] changes during EC coupling. Additionally, parallel experiments to those described in Aims 1-3 will be[unreadable] conducted in human myotubes generated from muscle samples of control individuals and MHS patients[unreadable] harboring analogous mutations in RyR1 (e.g. R163C and G2435R) to those used to make knock-in mice. For[unreadable] these experiments, human muscle samples collected by Core C from control individuals and patients of known[unreadable] genotypes and IVCT results will be used by Core B to propagate human myoblasts required for generating[unreadable] myotube cultures in Project 4. This project will combine the tools of molecular biology, mouse genetics,[unreadable] electrophysiology, confocal/electron microscopy, and high-speed Ca2+ imaging to asses the mechanisms by[unreadable] which MH/CCD mutations alter the function with the Ca2+ signaling triad.